Mx proteins, dynamin, VPS1~SPO15, and MGM1 comprise a new family of large GTPases (Mr=70,000 ~ 100,000) which are structurally conserved but have diverse biological activities. The alpha~beta interferon induced Mx proteins of vertebrates were originally identified as proteins that confer resistance to specific viruses. However, their antiviral activity may not be their primary function but a by~ product of an as yet unknown cellular activity. The Drosophila dynamin plays a role in endocytosis, whereas the function of vertebrate dynamin is unknown. The yeast VPS1~SPO15 protein is required for exocytosis and spindle pole body separation, and the yeast MGM1 protein for maintenance of mitochondrial DNA. Our studies focus on functional aspects of Mx proteins and dynamin. Current work with Mx proteins concerns their structure~function relationship. The cytoplasmic rat Mx2 and Mx3 proteins differ in several functional aspects although they are identical in all but 8 amino acids. Mx2 protein has potent anti~VSV activity, gives granular immunofluorescent staining, and binds microtubules in a GTP~dependent fashion. Mx3 protein has no measurable antiviral activity, gives diffuse staining, and does not bind microtubules in a GTP~dependent fashion. By taking advantage of the very similar primary structures of Mx2 and Mx3 proteins, we have identified in chimeric Mx2~Mx3 proteins a region close to the carboxy~terminus that is important for the antiviral activity and speckled appearance of Mx2. Interestingly, although the carboxy~terminal half of Mx2 alone was not sufficient for antiviral activity, it gave speckled staining. We are currently testing which region in Mx2 is involved in the GTP~dependent microtubule~binding activity. A different functionally important domain was identified by virtue of its ability to react with a monoclonal antibody that neutralizes the influenza virus resistance of interferon~treated mouse and rat cells, and the GTPase activity of purified rat Mx2 and Mx3 proteins. The identification of functionally important domains in Mx proteins may help us to elucidate the role Mx proteins play in interferon~treated cells and the mechanism by which they inhibit viruses. To genetically define the role dynamin plays in vertebrates, we have initiated a project to knock~out the mouse dynamin gene by homologous recombination in ES cells. We have isolated genomic DNA clones corresponding to the amino~terminal portion of the dyamin protein and are currently constructing a targeting vector to create a dynamin null allele.